Interplay among ZF-HD and GRF transcription factors during Arabidopsis leaf development

The growth-regulating factor (GRF) family of transcriptional factors are involved in the control of leaf size and senescence, inflorescence and root growth, grain size, and plant regeneration. However, there is limited information about the genes regulated by these transcriptional factors, which are in turn responsible for their functions. Using a meta-analysis approach, we identified genes encoding Arabidopsis (Arabidopsis thaliana) zinc-finger homeodomain (ZF-HD) transcriptional factors, as potential targets of the GRFs. We further showed that GRF3 binds to the promoter of one of the members of the ZF-HD family, HOMEOBOX PROTEIN 33 (HB33), and activates its transcription. Increased levels of HB33 led to different modifications in leaf cell number and size that were dependent on its expression levels. Furthermore, we found that expression of HB33 for an extended period during leaf development increased leaf longevity. To cope with the functional redundancy among ZF-HD family members, we generated a dominant repressor version of HB33, HB33-SRDX. Expression of HB33-SRDX from HB33 regulatory regions was seedling-lethal, revealing the importance of the ZF-HD family in plant development. Misexpression of HB33-SRDX in early leaf development caused a reduction in both cell size and number. Interestingly, the loss-of-function of HB33 in lines carrying a GRF3 allele insensitive to miR396 reverted the delay in leaf senescence characteristic of these plants. Our results revealed functions for ZF-HDs in leaf development and linked them to the GRF pathway.